Stability studies of amphetamine and ephedrine derivatives in urine

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Stability studies of amphetamine and ephedrine
derivatives in urine

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• Stability studies of
• amphetamine and ephedrine derivatives in urine
• By
• C. Jim enez , R. de la Torre
• M. Ventura ,J. Segura ,R. Ventura

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Introduction

• Knowledge of the stability of drugs in
  biological fluids is critical for proper
  interpretation of analytical results

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• thermal
• chemical
• degradation, matrix degradation
• metabolism, hydrolysis
• transport,
• handling or sample
• storage conditions

Losses of analys
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• Stability testing can be used

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Stability testing can be used

• explain discrepancies between reanalyses long
  after initial analyses
• determine time limits that must be imposed
  between the collection and analysis of samples
  for pharmacokinetic studies
• identify the optimal storage conditions

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DHHS Guidelines for Federal Workplace Drug
Testing(USA) 1998

• retain all confirmed drug positive urine
• least 1 year
• in frozen storage

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Introduction

• None psychostimulants like
• ephedrine derivatives.
• Ephedrine / amphetamine derivatives
• are included in the list of prohibited substances

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Sample

• ephedrine derivatives
• ephedrine, norephedrine, methylephedrine,
  pseudoephedrine, and norpseudoephedrine
• amphetamine derivatives
• (amphetamine, methamphetamine,
  3,4-methylenedioxyamphetamine (MDA),
• and 3,4-methylenedioxymethamphetamine (MDMA))

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Sample

• Sterile sample
• Long term
• Shot term
• Non Sterile sample
• (MDMA and methamphetamine)
• Long term
• Shot term

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Sterile samples

• Long-term stability
• 4 ?C and -20 ?C / at - 80 ?C as reference
• 1, 2, 3, 6, 10, 12, 18 and 24 months.
• Short-term stability
• 37 ?C / at -20 ?C for comparison purposes.
• 3 and 7 days.

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non Sterile samples

• Long-term stability
• 4 ?C and -20 ?C / at initial concentration as
  reference value for comparison
• 6 months
• Short-term stability
• 37 ?C / at -20 ?C for comparison purposes.
• 7 days.

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Preparation of Sterile samples

• Blank urine 2 ml sodium azide (0.1, w/v)
• clarified by filtration (three different filters)
• spiked standard
• distributed in aliquots under sterile conditions

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Preparation of non Sterile samples

• Blank urine 2 ml sodium azide (0.1, w/v)
• clarified by filtration (three different filters)
• spiked standard
• distributed in aliquots under sterile conditions
  in a laminar

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Samples analyzed

• Calibration samples were prepared in duplicate
• A control sample (three replicates) was analyzed
  in
• each analytical batch
• replicates of each aliquot of sample
• were analyzed at random in the analytical batch

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Precision and accuracy

• three different concentration
• three replicates of control urine samples
• Precision was expressed as the relative
  standarddeviation (R.S.D.)
• accuracy was expressed as the relative standard
  error (R.E.)

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Calculations

• The Dixons test (a 5)
• detect outliers in the replicates (n 5) of
  each aliquot
• ANOVA test (a 5)
• Homogeneity, adsorption of the analytes on the
  sterilizing filter and stability compare
  concentrations obtained at each storage condition
  with a reference value (concentration of aliquots
  of sample stored at the reference condition).

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Analysis of ephedrine derivatives
gas chromatograph (HP 5890 series II)
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Analysis of amphetamine derivatives

• HP 6890 series gas chromatograph
• HP 5973 mass selective detector

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• Results

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• coefficients (r2) up to 0.990
• amphetamine derivatives LOQ 71.0 ng/mL - 83.4
  ng/mL
• recoveries gt60 for amphetamine and MDA, and
  gt90 for methamphetamine and MDMA.

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Precision and accuracy

• Both methods good precision and accuracy
• lt20 for the low-concentration control urine
  samples
• lt15 for the medium and high concentration
  control urine samples.

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adsorption of the analytes on the sterilizing
filter
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2
4
1
0
not statistically significant (p gt 0.05) (data
not shown), indicating that all the sample
batches prepared for stability testing were
homogeneous.
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• Long-term stability

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-12 - 5
-10
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MDA 4 C 4
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• Shot -term stability

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• Long-term stability

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did not show a significant decrease (p lt 0.05)
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Discussion

• Moody et al.
• no significant change in analytes concentration
  for up to 17 months. Other studies have also
  demonstrated the stability of these drugs in
  non-preserved urine at different time and
  temperature conditions.

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Hughes et al.

• reported t he stability of amphetamine and
  methamphetamine in spiked urine samples stored at
  4 ?C for up to 6 months

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Discussion

• For long-term stability
• statistically significant only observed for the
  ephedrine derivatives at some of thestorage
  conditions tested
• not exceed the intra-assay precision of the
  corresponding analytical methods

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Dugan et al

• studied the stability in clinical samples tested
• before and after 1 year of storage at -20 ?C, 8

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Paul et al.

• investigated the effect of freezing (at -16 ?C to
  -18 ?C) on the concentration of amphetamine and
  methamphetamine in spiked urine samples stored
  for 45 days. In the same way, our observations
  are also in accordance with those obtained by

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Clauwaert et al.

• who demonstrated the stability of MDMA and MDA in
  non-preserved urine samples stored at -20 ?C, 4
  ?C and 20 ?C for 21 weeks.

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ephedrine and amphetamine derivatives

• sterile samples
• can be stored at the least. 4 ?C
• for up to 24 months for
• non-sterile samples
• can be stored at the least. 4 ?C
• for up to 6 months for

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Discussion

• The methodology presented when applied to other
  analytes
• may help to determine optimal storage conditions
  for urine samples
• to be used as reference materials and for
  positive urine
• samples that should be retained in drug-testing
  and antidoping
• control laboratories

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• The study demonstrates the feasibility of
• preparing certificate reference materials of
  successfully studied
• analytes. This is of special interest for those
  analytes for which a
• cut-off concentration has been established as
  positivity criterion
• for reporting adverse analytical findings, such
  as amphetamine
• derivatives in drugs of abuse testing, and
  ephedrine, methylephedrine
• and cathine in antidoping control.

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